Unravelling the effects of nano SiO2, nano TiO2 and their nanocomposites on Zea mays L. growth and soil health

Amidst the challenges posed by climate change, exploring advanced technologies like nanotechnology is crucial for enhancing agricultural productivity and food security. Consequently, this study investigated the impact of nano SiO2 (nSiO2), nano TiO2 (nTiO2) and SiO2/TiO2 nanocomposites (NCs) on 30-day-old Zea mays L. plants and soil health at concentrations of 100 and 200 ppm. Results showed that nSiO2 and nTiO2 at 100 ppm and SiO2/TiO2 NCs at both concentrations, positively influenced plant growth, with the best stimulation observed at 200 ppm of SiO2/TiO2 NCs. Improved plant growth was associated with higher chlorophyll content, photosynthetic rate, transpiration rate, stomatal conductance, rhizospheric N-fixing and phosphate solubilizing bacterial population and plant nutrient uptake. Additionally, treated plants exhibited increased cellulose and starch levels. Malondialdehyde (MDA) content was lower or similar to that of the control, except at 200 ppm of nTiO2-treated shoots. Antioxidant enzyme activities fluctuated, indicating physiological adjustments. Overall, 100 ppm of nTiO2 as well as nSiO2 and 100 and 200 ppm of SiO2/TiO2 NCs improved soil fertility and Z. mays growth, suggesting potential benefits for sustainable agriculture. The findings lay the foundation for more comprehensive investigations into the long-term fate of nanomaterials in soil and their intricate molecular-level interactions with Z. mays.

The use of nanocomposites (NCs) made by combining metals with other inorganic or organic materials has gained attention because of their superior qualities over individual nanoparticles 19 .An NC of nSiO 2 and nTiO 2 is expected to exhibit synergistic effects and improved performance as it will combine the biocompatible highly porous structure and large specific surface area of nSiO 2 with the photocatalytic activity of nTiO 2 .Adding positively charged nTiO 2 to nSiO 2 may also limit excessive chelation of other nutrients by Si while providing adequate positive surface charge to improve interactions with plant cells.Moreover, the complexation of nTiO 2 with nSiO 2 will decrease the TiO 2 fraction available to produce hazardous metal ions and hence damaging ROS.
Though the effects of nSiO 2 and nTiO 2 on Z. mays have been the subject of multiple research, the findings have been equivocal and there are still significant gaps in our understanding at biochemical and physiological levels.Furthermore, the impact of SiO 2 /TiO 2 NCs on Z. mays has not yet been investigated.Therefore, the current work assesses the morphological, biochemical, and physiological changes in 30-day-old Z. mays plants cultivated in nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs spiked soil.Additionally, the effects of nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs on plant nutrient uptake and soil microflora have also been tested as a marker of soil health.

Synthesis of nSiO 2
The Stober technique 20 was used to synthesize nSiO 2 .To make nSiO 2 , tetraethoxysilane (TEOS) (11.2 mL) was added to a mixture of 10 mL ethanol and 35 mL deionized water and stirred for 10 min.After that, some drops of hydrochloric acid (HCl) (1 M) were added and the solution was stirred magnetically (Tarsons-SPINOT DIGI-TAL, MC02, India) for 50 min at 60 °C until it became a homogenous white translucent solution.The prepared solution was left at 25 °C for 2 h to facilitate gel formation.After 6 h of drying at 90 °C in an oven, crystalline SiO 2 particles were ground to yield a white powder of nSiO 2 .

Synthesis of nTiO 2
The nTiO 2 were prepared by the procedure followed by Ghows and Entezari 21 .A mixture of glacial acetic acid (0.2 mL) and distilled water (50 mL) was sonicated (Q-Sonica Sonicators, Q125, U.S.A.) for 10 min.To it, a solution of 2 mL titanium tetra-isopropoxide (TTIP) and 5 mL ethanol was added dropwise and sonicated at 25 °C for 3 h (20 kHz,125 W, 70% amplitude).After centrifuging (Plasto Crafts, Rota 4R-V/Fm, India) the solution at 17,608×g for 20 min, the precipitates were repeatedly cleaned with distilled water and ethanol.The product was dried for 6 h at 40 °C.

Synthesis of SiO 2 /TiO 2 NCs
TEOS and TTIP were selected as the sources of silica and titania, respectively 22 .NCs were synthesized in two steps.In the first step, under constant stirring, TEOS was added dropwise to the methanol and both were combined in 1:6 ratios to synthesize nSiO 2 .Then, 0.05 mol (1.52 mL) HCl was added dropwise to the solution to maintain a pH of 2.0.The solution was stirred for around 2 h to get a homogenous solution.In the second step, TTIP and distilled water were mixed in a 1:14 ratio until a homogenous TiO 2 solution was formed.Then, TiO 2 and SiO 2 were mixed in a 1:1 ratio under continuous stirring until a homogenous solution was formed and to it, 0.05 mol (2.2 mL) NH 4 OH solution was added dropwise.Finally, the mixture was stirred for 10 min and dried at 90 °C to get the SiO 2 /TiO 2 NCs powder.

Experimental design
The seeds of Z. mays (HQPM-7) were purchased from the Regional research centre of Haryana Agriculture University at Uchani, Karnal.The seeds were procured and used by following all the relevant guidelines.They were immersed in a 0.1% mercuric chloride solution for 2 min to ensure surface sterilization and rinsed at least 5 times with distilled water.Fertile farm soil was collected from the Research Centre, MDU, Rohtak and air-dried before experimental use.Soil analysis by ICP-MS (ICAP 600 Series, ICP Spectrometer from Thermo Scientific) at Soil Testing Lab, Rohtak revealed that it had the following properties: Electrical conductivity 0.23 dS m −1 , organic carbon 0.54%, pH 7.02, Zn 3.14 ppm, Fe 19.54 ppm, Mn 4.04 ppm, S 15.32 ppm, K 65 ppm and P 14 ppm.
A total of seven experimental groups were raised in pots: one control group that had no nanomaterial at all and six treatment groups, corresponding to plants raised in soil spiked with either nSiO 2 or nTiO 2 or SiO 2 /TiO 2 NCs at two different concentrations i.e., 100 and 200 ppm, respectively.Each group had five replicates with five plants per replicate, making a total of 25 plants per experimental group.The emergence of the radicle was considered an indicator of seed germination.The plants were grown at the average day/night temperature of 37.0/21.8°C.They received photosynthetic photon flux density of 1480 to 1850 µmol m 2 s −1 light intensity on a clear, bright sunny day in April/May, having approximately 12 h light and 12 h dark period.
Whole plants were carefully removed from the soil after 30 days.Rhizospheric soil samples were collected from plant roots and the plants were carefully washed with distilled water.The roots and shoots were separated and their length, fresh and dry weight were measured.The number of axial roots was also counted.For further investigation, the plants were crushed in liquid nitrogen and stored at − 20 °C.The collected rhizospheric soil was air dried and utilized for checking the plant growth-promoting bacteria by serial dilution method.

Mineral and metal uptake by Z. mays plants
The shoots and roots of the harvested plant samples were dried at 80 °C for 48 h to examine the amount of mineral nutrients (N, P, K and Mg) taken up by control and treated Z. mays plants 23 .The contents of Si and Ti in dried plant samples were also analyzed by Energy Dispersive Spectroscopy (EDS, Hitachi: 8010) at SAIF, Punjab University, Chandigarh.

Chlorophyll and protein content
The determination of chlorophyll contents was based on the method of Thakur et al. 24 .Protein contents of the root and shoots were measured by the Bradford method 25 using the standard curve of BSA (10-100 mg g −1 ).The results for both chlorophyll and protein were expressed as mg g −1 of plant material.

Starch and cellulose content
To measure starch content, the plant sample (0.1 g) was dissolved in 10 mL hot ethanol (80%) to remove all the sugars and then 6.5 mL perchloric acid (52%) was added to convert starch to glucose units 26 .The amount of glucose was estimated by the phenol method.To determine cellulose content, the plant sample (0.1 g) was digested in 6 mL of nitric acid.After digestion, all sugars were removed except cellulose and further, anthrone assay was used for cellulose quantification 27 .Starch and cellulose contents were calculated using a glucose standard curve (10-100 µg mL −1 ) and results were represented in mg g −1 of plant material.

Gas exchange parameters
In 30-day-old nanomaterials treated and control plants, gas exchange parameters such as net photosynthesis (A), stomatal conductance (G) and transpiration rate (E) were measured with a handheld gas exchange device (LCi-SD, ADC BioScientific Ltd., UK).Observations were made on fully inflated leaves between 10:00 and 12:00 A.M. at 26 ± 0.1 °C.At the time of measurement, CO 2 concentration was 300 ppm and the photon flux density was 1250 ± 10 µmol m −2 s −128 .

Lipid peroxidation and antioxidant enzymes activities
Heath and Packers 29 method was used to calculate the amount of MDA produced by lipid peroxidation, which was reported as µM MDA g −1 fresh weight.The present work investigated the nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs-induced modulations in the activities of four antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx), and glutathione peroxidase (GPx).To accomplish this, 0.5 g of plant tissue was homogenized in 5 mL of potassium phosphate buffer (0.1 M, pH 7.0) and subsequently cold centrifuged for 10 min at 17,608×g.The resulting supernatant served as the enzyme source for subsequent experiments.In all experiments, potassium phosphate buffer (0.1 M, pH 7.0) was used as the blank.
SODs are metalloproteins that catalyze the dismutation reaction of superoxide to O 2 and H 2 O 2. This assay was performed by NBT (nitroblue tetrazolium) reduction method 30 31 and the unit activity of CAT was expressed as U mg −1 min −1 .APx activity was calculated by detecting the decrease of A 290 nm due to oxidation of ascorbate by H 2 O 2 32 .The activity of GPx was directly related to the quantity of oxidized glutathione and was assessed using the procedure described by Rani et al. 33 .

Influence on soil microorganisms
The effects of nanomaterials on soil microbes were determined by culturing N-fixers and phosphate solubilizers on their specific media.Azotobacter and Azospirillum are free-living, gram-negative and aerobic N-fixers that were isolated using Ashby's mannitol media and Azospirillum medium with 0.17% agar, respectively.Pikovaskay's growth media was used to check the growth of phosphate solubilizers.

Statistical analysis
A completely randomized block design was used in the experiment.The experimental data was analyzed using IBM SPSS Statistics 26 software for one-way ANOVA and a post-hoc analysis with the Tukey test.Different letters represent significant differences at p ≤ 0.05.Origin 8 (Origin Lab Corporation, United States) was used to prepare graphs.Standard error bars in the graph indicate the variability of data.

Results and discussion
Characterization FESEM images of the nSiO 2 (Fig. 1A) and nTiO 2 (Fig. 1B) indicated that they were spherical to polygonal in shape.The nSiO 2 were well dispersed, while the nTiO 2 showed a propensity to aggregate.The network of irregular spheres is shown to be interconnected and grouped in the SiO 2 /TiO 2 NCs micrograph shown in Fig. 1C.The TEM picture of nSiO 2 , as seen in Fig. 2A, revealed that the size of the nanoparticles ranged between 5 and 10 nm.The nTiO 2 were clustered with an average size of 20-30 nm (Fig. 2B), whereas in Fig. 2C, the TEM image of SiO 2 / TiO 2 NCs indicated that the size of NCs was between 30 and 40 nm.
The hydrodynamic diameter of nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs suspension as determined by Zetasizer analyzer was 256.6 ± 78.36 nm, 369.1 ± 138.7 nm and 375.5 ± 145.8 nm and zeta potential values were − 25.4 ± 4.10, 38.2 ± 4.96 and − 4.09 ± 3.63 mV respectively Supplementary Fig. 1).According to zeta potential values, nSiO 2 were stable, nTiO 2 were highly stable and NCs were unstable.The positive charge of nTiO 2 decreased the negative  www.nature.com/scientificreports/charge of nSiO 2 after combining with it.The presence of a negative charge on the NCs surface suggests that negatively charged nSiO 2 were present on the surface and nTiO 2 was present in the core of the NCs.
The size distribution of nanoparticles can be explained by the polydispersity index (PDI) value.The PDI values for the nSiO 2 , nTiO 2 , and SiO 2 /TiO 2 NCs suspension in the current investigation were 0.54 ± 0.02 (highly polydispersed), 0.208 ± 0.01 (moderately dispersed) and 0.355 ± 0.03 (moderately dispersed), respectively.Results indicated that the moderately dispersed suspension produced by the NCs had a better size distribution than that of the highly polydisperse nSiO 2 suspension.
The XRD pattern of nSiO 2 , nTiO 2 and the NCs is given in Fig. 3.The XRD pattern of nSiO 2 showed the presence of a very broad peak displayed around 18-25 2Ѳ (Fig. 3A).This broad peak confirmed the small size and amorphous nature of the synthesized nanoparticles 34 .The nSiO 2 did not contain any additional impurity peaks.XRD pattern of nTiO 2 in Fig. 3B, showed sharp peaks at 25, 38, 47, 54 and 62 thus confirming the crystalline phase of nTiO 2 , which is quite similar to the nTiO 2 reference pattern (JCPDS 21-1272).The broad peak visible in Fig. 3C, which is located around 20-30 2Ѳ, indicated that the SiO 2 /TiO 2 NCs were amorphous in character.Xie et al. 35 reported that the addition of a small amount of nSiO 2 into pure nTiO 2 inhibits the growth of crystalline structure.

Soil analysis
Electrical conductivity (EC = 0.23 dS m −1 ), organic carbon (OC = 0.54%), pH (7.02, Neutral) and nutrients: Zn (3.14 ppm), Fe (19.54 ppm), Mn (4.04 ppm), S (15.32 ppm), K (65 ppm) and P (14 ppm) present in soil were analyzed by ICP-MS at Soil Testing Lab, Rohtak.All the nutrients were present in the soil in between the quantity suggested by the agriculture and farmer welfare department report, Haryana.According to the soil analysis results, the soil quality was good for conducting the experiments.Soil organic matter, in particular, has the potential to alter NP mobility and bioavailability by influencing their aggregation state, surface charges, polarization state, zeta potential and intermolecular interactions 36 .

Quantification of nanomaterials
Dried plant samples were analyzed by EDX for quantification of nanoparticles accumulated by them.Percent accumulation of Si in control and nSiO 2 -treated plants at 100 and 200 ppm concentration was found to be 0.32% in control and 0.93 and 1.27%, respectively (Fig. 4A).In the roots, too, percent Si accumulation increased from 1.22 in control to 2.39 and 3.54% at 100 and 200 ppm, respectively.Suriyaprabha et al. 37 also reported that 10 and 15 kg ha −1 nSiO 2 increased Si accumulation in maize plants by 0.57 and 0.82%, respectively.In SiO 2 /TiO 2 NCs treated plants, there was approximately 4-5 times more percent accumulation of Si in the shoots (i.e.4.22% at 100 ppm and 4.82% at 200 ppm concentration) compared to the control, while the percent accumulation of Si in the roots of NCs was 2.36% at 100 ppm and 2.87% at 200 ppm concentration.When plants were treated with nSiO 2 , the accumulation of Si in the roots was higher compared to the shoot, whereas for NCs treated plants, the shoots accumulated more Si than the roots.Si is abundant in the soil and the charge on nanoparticles, plant roots, cellular components, biomolecules and cell wall influences its uptake and transportation in plants.The nSiO 2 having higher negative charge intensity is not well retained by the negatively charged cell wall; instead, it interacts strongly with other charged plant cell constituents like proteins and nucleic acids, which may impede its passage through plasmodesmata and limit its ability to migrate from one cell to another.Furthermore, the Casparian strip also prevents negatively charged nSiO 2 from moving radially 38,39 .Conversely, the presence of fewer negative charges on the surface of NCs in comparison to nSiO 2 may have facilitated their symplastic transportation to the central stele and subsequent translocation to the above-ground parts of the plant 40,41 .Additionally, in SiO 2 /TiO 2 NCs, nTiO 2 interacts with the nSiO 2 , imparting it a hydrophilic character that may favour their adsorptive behaviour in soil 42 .Titanium (Ti) accumulation in the shoots of Z. mays plants increased from 0.02% in the control to 0.18 and 0.20% in the nTiO 2 treatments at 100 and 200 ppm concentrations, respectively (Fig. 4B).Ti accumulation in the roots increased from 0.02% in the control to 0.14% and 0.18% at 100 and 200 ppm, respectively.Similar Ti accumulation was found by Thakur et al. 24 in mung bean plants treated with nTiO 2 at doses of 10 and 100 mg L −1 .As shown in Fig. 4B, the percent accumulation of Ti in the shoots was higher in NCs (0.26% at 100 and 0.31% at 200 ppm concentration) than in nTiO 2 .The matrix composition, which includes the amount of organic matter, free ions and pH, influences TiO 2 mobility in soils 43 .Additionally, changes in the surface area, zeta potential and surface affinity may all affect TiO 2 availability in soil and plants 44 .
In the nTiO 2 -treated plants, the accumulation of Ti in the shoots was lower compared to the Ti content in the shoots of NC-treated plants.Once the metal ion is absorbed, its accumulation in the shoots is directly related to the ability of plants to transport it from cell to cell.Because of the higher positive charge on the nTiO 2 , they might have interacted more strongly with the negatively charged cell wall and other biomolecules in the cytoplasm, hindering their movement both through the apoplast and symplast and hence their accumulation in the shoots.

Effect on growth parameters
The effect of nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs on the growth parameters of Z. mays after 30 days of growth is presented in Table 1.The shoot length of nSiO 2-treated plants was comparable to control plants at 100 ppm while it decreased by 12% at 200 ppm.The root length increased by 20% at 100 ppm of nSiO 2 and decreased marginally by 4% at 200 ppm.Both concentrations of nSiO 2 increased parameters such as root and shoot number, as well as fresh and dry weight, however, the stimulation was higher at 100 ppm compared to 200 ppm.The fresh weight of the shoot and root increased by 9 and 41% and the dry weight by 49 and 5% at 100 ppm, respectively.At 200 ppm of nSiO 2 , the fresh and dry weight of the shoot and root increased slightly and were largely comparable to the control plants.nTiO 2 at 100 ppm stimulated shoot growth, with a slight increase of 2% in shoot length, 5% fresh weight and a substantial increase of 52% in dry weight, respectively.However, a slight inhibition of shoot growth was observed at 200 ppm, with decreases of 4%, 10%, and 24% in shoot length, fresh weight, and dry weight, respectively.Contrary to this, both 100 and 200 ppm concentrations of nTiO 2 stimulated root growth, however, the growth at 200 ppm was lower compared to 100 ppm concentration.The root length increased by   45 and Liu and Lal 46 also reported that nSiO 2 increased germination percentage, lateral root development, and plant biomass in rice plants.Though nSiO 2 is largely associated with improved plant growth, at higher concentrations it may potentially interfere with the absorption of other essential nutrients including N and P 47 .Slightly reduced shoot and root length by nSiO 2 at 200 ppm could possibly be due to reduced absorption of N and P by the roots of these plants (Table 3).
The nTiO 2 may promote cell expansion and plant growth by loosening the cell wall and increasing membrane fluidity 48 .Growth stimulation by nTiO 2 is also in part due to the enhanced ability of plants to absorb nutrients from the soil 49 , as observed in the present study (Table 3).Nonetheless, at higher concentrations, nTiO 2 also produces damaging ROS, which inhibits the growth of plants.Stimulatory effects of nTiO 2 (100 mg L −1 ) on roots were previously demonstrated in wheat 49 and in chickpea 50 plants.Zahra et al. 51 reported that root growth and development was influenced by the absorption of nutrients like K, N, Mg and P by the plants in the presence of nTiO 2 .Raliya et al. 52 also concluded that nTiO 2 -induced uptake of mineral elements in the plants led to increased growth and biomass of mung bean plants.In another study, it was observed that nTiO 2 had a positive impact on wheat at concentrations up to 60 mg kg −1 , but at higher levels (100 mg kg −1 ), it had an adverse effect on wheat roots 53 .
Enhanced growth by the NCs might be due to increased uptake of N, P and K by the SiO 2 /TiO 2 NCs treated plants (Table 3) which besides providing essential micronutrients also enhance their abilities to absorb and utilize water resulting in enhanced growth.

Photosynthetic parameters
Total chlorophyll content, photosynthetic rate, transpiration rate and stomatal conductance in 30 days old Z. mays plants at both 100 and 200 ppm of nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs are presented in Table 2. Chlorophyll content increased by 75 and 29% in nSiO 2 treated plants, 66 and 79% in nTiO 2 treated plants and 34 and 81% in NCs treated plants at 100 and 200 ppm concentrations, respectively.The percentage increase in transpiration rate was 68, 24 and 87% at 100 ppm and 4, 101 and 141% at 200 ppm of nSiO 2 , nTiO 2 and NCs, respectively.Table 1.Effect of nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs on the growth of 30-day-old Z. mays plants.Data is presented as mean ± S.D (n = 5), with different letters indicating significant differences at p < 0.05.Though, the values of all photosynthetic parameters significantly increased in all treatment groups compared to the control, however, the best stimulation was observed at 200 ppm of SiO 2 /TiO 2 NCs.This suggests that NCs facilitated increased translocation of Si and Ti to the leaves might have enhanced the photosynthetic capacity of the Z. mays plants.
Suriyaprabha et al. 37 reported that nSiO 2 led to an increase in Si accumulation, enhancing the photosynthetic activity of Z. mays plants by enhancing the leaf surface area.This, in turn, improved light absorption and the photosynthetic activity of both chlorophyll a and b.Similar findings were documented for nTiO 2 treatment on wheat plants by Shafea et al. 54 .A study on wheat cultivars soaked in various concentrations of nTiO 2 (0.025, 0.05, 0.1, 0.2, and 0.5%) revealed that 0.1% nTiO 2 significantly improved seed potential by raising vigour index, plant height and root length, shoot and root dry weight, fresh matter and the composition of photosynthetic pigments such as chlorophyll.Furthermore, nTiO 2 have been demonstrated to improve plant uptake of many mineral nutrients, including Ca, Mg and K, which improve photosynthetic efficiency by enhancing chlorophyll synthesis and gas exchange 55 .A study conducted by Yang et al. 56 revealed that in spinach, nTiO 2 promotes the organic N content of the plant such as the amount of chlorophyll and proteins by increasing the absorption of inorganic N from the soil.
Carbohydrates are formed in plants by photosynthesis and make up about 70% of solid plant material.Additionally, Z. mays is a rich source of cellulose, a type of carbohydrate that directly influences growth, mechanical support, the conversion of biomass into fuel and the development of other biomass-based products 57 .Cellulose content was increased by both the nanoparticles and their NCs in both root and shoot at all the concentrations, which correlates well with enhanced chlorophyll content and photosynthetic rate 58 .The percentage increase of cellulose in shoots of treated plants was 124 and 196% for nSiO 2 , 56 and 88% for nTiO 2 and 23 and 196% for NCs at 100 and 200 ppm concentrations, respectively.In roots, the percentage increase was 32% and 21% for nSiO 2 , 11 and 120% for nTiO 2 and 2 and 42% for NCs at 100 and 200 ppm concentrations, respectively.
Starch content showed significant enhancement across all treatments.At 100 ppm, there was an approximate increase of 42, 25 and 15% in shoots treated with nSiO 2 , nTiO 2 and NCs, respectively.Similarly, at 200 ppm, the increase was approximately 16, 20 and 47% for the respective treatments.In roots, there was about a 48% increase at 100 ppm for both nSiO 2 and nTiO 2 , with a marginal 3% increase for NCs.At 200 ppm concentration, the root starch content increased by 5, 35 and 72% after treatments with nSiO 2 , nTiO 2 and NCs, respectively (Table 2).The starch content in both the shoots and roots of Z. mays was higher at a 100 ppm concentration of both nSiO 2 and nTiO 2 compared to the content at 200 ppm of these nanomaterials.However, there was an increase in starch content with the rise in concentration of SiO 2 /TiO 2 NCs.The increase in starch and cellulose content might be a direct consequence of the enhanced photosynthetic efficiency of the treated plants.

Protein content and antioxidant status
Compared to the control group, both nanomaterials and NCs significantly increased protein content in both roots and shoots, with a more pronounced effect observed at 200 ppm compared to 100 ppm (Fig. 5A).Specifically, protein content increased by 66 and 143% in shoots and by 10 and 37% in roots at 100 and 200 ppm of nSiO 2 , respectively.For nTiO 2 , protein content increased by 92 and 174% in shoots and by 20 and 55% in roots at 100 and 200 ppm concentrations, respectively.Similarly, NCs led to a percentage increase of 31 and 13% at 100 ppm and 154 and 20% at 200 ppm concentration in shoots and roots of treated plants, respectively.Nanomaterials may modulate plant metabolism to trigger the synthesis of proteins involved in growth, development and stress responses 59 .Suriyaprabha et al. 37 reported higher protein contents in maize following nSiO 2 treatment.According to Yang et al. 56 , nTiO 2 enhanced the absorption of inorganic N, as well as increased the activity of enzymes that help plants to synthesise protein and chlorophyll.
On the 30th day, the content of MDA in the shoots of all treated plants was lower than the control, except for nTiO 2 at 200 ppm, where a 10% increase was observed (Fig. 5B).However, no appreciable difference in the MDA content of treated roots was observed.Reduced lipid peroxidation, which damages lipids and proteins in cells, is expected to be the source of higher protein concentrations in the treated plants.However, despite experiencing oxidative stress, the plants had the highest protein content at 200 ppm of nTiO 2, which correlate well with enhanced activities of antioxidant enzymes (SOD, CAT and GPx).Enhanced protein content is considered an adaptive response where the plants increase the synthesis of certain stress-related proteins involved in ROS detoxification 60 .
The nSiO 2 are also well known for their ability to reduce oxidative stress, potentially through the scavenging of ROS and the upregulation of antioxidant enzyme activities (Fig. 5C-F).In the present work, nSiO 2 raised the activities of shoot SOD (Fig. 5C) and GPx (Fig. 5E) at both 100 and 200 ppm.The CAT (Fig. 5D) and APx (Fig. 5F) activities decreased in shoots as well as in roots at both concentrations.Torabi et al. 61 observed similar outcomes in Borago officinalis plants grown in the presence of Si and under salt stress.The authors observed that at 1.5 mM Si, the MDA levels decreased by 38%, whereas SOD activity increased significantly while the activity of CAT and APx remained unchanged.
The nTiO 2 did not affect the shoot SOD and GPx activities but decreased the CAT activity at 100 ppm concentration, which correlated with a decrease in MDA content in the shoot of Z. mays plants, but at 200 ppm concentration of nTiO 2 , a slight increase in SOD, CAT and GPx activities and MDA content was observed.It indicated that nTiO 2 developed stress at 200 ppm concentration in the shoot of Z. mays plants which was also evident from slightly reduced shoot growth at this concentration.
In roots, activities of all the antioxidant enzymes i.e., SOD, CAT, APx and GPx decreased at both the concentrations of nTiO 2 which indicated that they did not harm the roots of Z. mays plants.
In the shoots of SiO 2 /TiO 2 NCs treated Z. mays plants, SOD activity increased, APx activity decreased and GPx activity was not affected at both 100 and 200 ppm of NCs.The CAT activity increased at 100 ppm but decreased at 200 ppm of NCs.In roots, SiO 2 /TiO 2 NCs decreased SOD and CAT activities and increased APx and GPx activities at both concentrations.Numerous factors, including the type and surface charge of the nanomaterial, its concentration, the duration of exposure and the specific plant species, might affect the intricate interaction between nanomaterials and antioxidant enzymes.Studies have revealed that the effects of nanomaterials on the activity of enzymes are inconsistent and unpredictable.For instance, nTiO 2 enhanced the GPx, CAT, and SOD activities in Lemna minor 62 and decreased the GR and APx activities in Vicia faba 63 .However, nTiO 2 increased the activities of CAT, SOD, GPx, and APx in spinach 64 .Consequently, it remains challenging to make generalizations about the effects of nanomaterials on antioxidant enzymes.

Influence on soil microorganisms and uptake of nutrients by plants
Plant productivity is highly influenced by soil microbes 65 .The impact of nanomaterials on plant developmentpromoting microorganisms, such as N-fixers and phosphate solubilizers, was investigated in the current study (Fig. 6).Phosphate-solubilizing bacteria (PSB) can convert insoluble organic and inorganic P to soluble P that plants can easily absorb.P plays an important role in plant growth and development, N fixation and conversion of sugar to starch 66 .nSiO 2 and nTiO 2 treatment increased the population of PSB at both 100 and 200 ppm concentrations (Fig. 6).Due to the improved PSB count in the presence of both nanoparticles, higher P content in all the treated Z. mays plants was observed, except for reduced P content in the roots of nSiO 2 treated plants (Table 3).While Si normally increases the bioavailability of nutrients in the soil, occasionally, particularly at higher concentrations and neutral pH, Si may immobilize P by forming complexes with phosphate ions or it may compete with P for uptake by plant roots 67 .When the soil was amended with SiO 2 /TiO 2 NCs, PSB count was almost three times the control and much higher than recorded for the individual nanoparticles.Solubilization of phosphate by microorganisms correlated well with the uptake of P by Z. mays plants as given in Table 3.
Significant increase in the colonies of N-fixing bacteria, Azospirillum was observed at 100 ppm concentration of nSiO 2 but the same decreased at 200 ppm of nSiO 2 , though the decrease was insignificant.The Azospirillum colonies increased at both concentrations of nTiO 2 as compared to control but the enhancement was more at 100 ppm compared to 200 ppm.In the case of NCs, Azospirillum count increased in a concentration-dependent manner.The count of another N-fixing bacteria, Azotobacter increased after the nSiO 2 treatment at 100 ppm but decreased significantly at 200 ppm.Azotobacter population increased in the presence of nTiO 2 and NCs at both 100 and 200 ppm; the highest was at 200 ppm of NCs, which corresponded to the highest N uptake by plants treated with 200 ppm of SiO 2 /TiO 2 NCs.Although the count of N-fixing bacteria increased at 200 ppm nTiO 2 , there was an approximately 8% decrease in the N content of the shoot compared to control plants.This discrepancy may be attributed to the generation of oxidative stress by nTiO 2 at 200 ppm, which can influence N assimilation, storage and translocation within the plant potentially leading to changes in shoot N content.The combination of oxidative stress and low nitrogen content resulted in reduced shoot growth in the treated plants.For the rest of the treatment groups, an increase in the colonies of both N fixers correlated well with the data on N uptake by Z. mays plants.The nSiO 2 at 200 ppm was not so conducive to the N fixers and the plants had the reduced N and lowest P contents amongst all the treated plants, whereas the NCs of SiO 2 with TiO 2 yield the highest PSB and Azotobacter activities corresponding to greater accumulation of N and P by plant shoot and roots.
Potassium (K) ranks as the third most important macronutrient, following N and P, required to enhance plant productivity.K enhances the utilization efficiency of N, which, in turn, is directly related to plant growth.Mg is another important micronutrient that plays a very important role in chlorophyll synthesis, carbon fixation and as a cofactor of various enzymes.Compared to the control plants, uptake of K and Mg by Z. mays plants increased in all treatment groups.The increase in the concentration of Mg was also supported by the increase in chlorophyll and carbohydrate content.The nanomaterials have greater surface area for mineral adsorption and reduce nutrient leaching which may enhance the bioavailability of mineral nutrients.Mali and Aert 68 observed that in the presence of Si in the soil, nutrients like N, P and K were more available to the plants 69 .The nTiO 2 may also improve the cation exchange capacity of the soil and help retain the nutrients in the root zone for plant uptake.Increased root growth in the presence of SiO 2 /TiO 2 NCs along with their unique surface properties and catalytic activity may have promoted nutrient retention and transport in the rhizosphere, improving their uptake by plants.

Conclusion
Considering the substantial impact of nanomaterials and NCs on plant growth and soil health, the effect of adding nSiO 2 , nTiO 2 and SiO 2 /TiO 2 NCs to the soil at 100 and 200 ppm concentrations has been described on Z. mays health, soil microbial activity and nutrient uptake by plants (Fig. 7).The results on plant growth, chlorophyll levels, photosynthetic and transpiration rate, stomatal conductance, as well as protein, carbohydrates and MDA levels, soil microbial activity and plant nutrient (N, P, Mg and K) uptake along with changes in the activities of antioxidant enzymes, suggest that these nanomaterials can be safely used to boost soil fertility and support Z. mays growth.However, nSiO 2 and nTiO 2 at 200 ppm concentration had adverse effects.nSiO 2 at 200 ppm decreased shoot and root length, the count of N-fixing bacteria, N uptake in both shoots and roots and P uptake in roots, while increasing the root MDA contents.Decreased shoot growth in plants exposed to 200 ppm of nTiO 2 was attributed to reduced N contents in the shoots and oxidative stress induced by the nanoparticles.In contrast, SiO 2 /TiO 2 NCs at 200 ppm resulted in the maximum stimulation of plant growth, surpassing both nSiO 2 and nTiO 2 used individually at the same concentration, highlighting the superiority of NCs.The main findings of the study are given in Fig. 7.
Nevertheless, there is a need for further research on the fate, behaviour and transformation of nanomaterials in soil, their interaction with soil organic matter and their effects on other soil microorganisms.Additionally, the intricate molecular-level interactions of nanomaterials with plants remain an evolving area of research and a comprehensive understanding of these interactions is essential for the sustainable use of nanomaterials in agriculture.

Figure 4 .
Figure 4. Accumulation of Si (A) and Ti (B) in the shoots and roots of 30-day-old Z. mays plants, as determined by EDS.Data represents the mean of five replicates, with standard error bars indicating the variability of data.

FFigure 5 .
Figure 5.Protein (A) and MDA (B) contents, as well as the activities of SOD (C), CAT (D), GPx (E) and APx (F) in the shoots and roots of Z. mays treated with 100 and 200 ppm of nSiO 2 , nTiO 2 and TiO 2 /SiO 2 NCs.Data represents the mean of five replicates, with different letters denoting significant differences at p ≤ 0.05.Standard error bars indicate the variability of data.

Figure 7 .
Photosynthetic pigmentsProtein content Carbohydrate . CAT activity was proportional to a decrease in Vol:.(1234567890) Scientific Reports | (2024) 14:13996 | https://doi.org/10.1038/s41598-024-61456-x and 4%, root fresh weight by 28 and 12% and root dry weight by 22 and 8% at 100 and 200 ppm of nTiO 2 .As evident from Table1, NCs of SiO 2 /TiO 2 increased all the parameters in a concentration-dependent manner in Z. mays plants.NCs increased the shoot length by 2 and 6%, root length by 3 and 23%, shoot fresh weight by 9 and 12%, root fresh weight by 1 and 50%, shoot dry weight by 7 and 31% and root dry weight by 2 and 5% at 100 and 200 ppm respectively.The nSiO 2 promotes root mass and development, enabling plants to penetrate the soil and facilitating the absorption of water and nutrients.Liu et al.